Monoclonal antibodies and cocktails for treatment of ebola infections

ABSTRACT

Described herein are compositions and methods for the prevention and treatment of ebolavirus infection. In certain embodiments of the present invention, monoclonal antibodies substantially similar to those described herein, as well as affinity matured variants thereof, alone or in combination, provide therapeutic efficacy in a patient against multiple species of ebolavirus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/898,524, filed Feb. 17, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/460,200, filed Feb. 17, 2017, eachof which is incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under U19 AI109762awarded by NIH and HDTRA-13-C-0018 awarded by DTRA. The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference. Said ASCII copy, created on May 20, 2020, is named1123-2007-ST25 and is 34,674 bytes in size.

BACKGROUND

Ebolaviruses are members of the family Filoviridae which infect humansand non-human primates (NHPs) causing hemorrhagic fever with mortalityrates up to 90%. Ebolaviruses include Ebola virus (EBOV), Sudan virus(SUDV), Bundibugyo virus (BDBV), Reston virus (RESTV), and Taï Forestvirus (TAFV), which are causative agents of the hemorrhagic fever [1,2]. A summary of the ebolaviruses can be found in Burk, et. al.,Neglected Filoviruses. FEMS Microbiology Reviews, 40, 494-519 (May,2016), and the differences between the viruses have been wellcharacterized and well known in the art. Between 1967 and 2013, 31filovirus disease outbreaks have occurred, mainly in central Africa witharound 2,000 confirmed cases. Of these 31 outbreaks, 16 were caused byEBOV. The unprecedented 2013-2016 Ebola virus disease epidemic led tomore than 27,000 cases and 11,100 deaths in the first 14 months. Thereare currently no approved treatments or vaccines for filoviruses, andmost advanced experimental treatments focus only on EBOV. Given thatother filoviruses have caused sizeable outbreaks broadly protectivetreatment options are urgently needed.

Several studies have shown that filovirus glycoprotein (GP)-specificneutralizing antibodies (nAbs) can reduce mortality followingexperimental inoculation of animals with a lethal dose of EBOV [3-9].The primary target of these neutralizing antibodies, the filovirussurface GP, is a trimer composed of three heavily glycosylated GP1-GP2heterodimers. The GP1 subunit can be divided further into base, head,glycan cap and mucin-like domains [10]. During viral entry, themucin-like domain and glycan cap mediate binding to multiple hostattachment factors present on the cell membrane. After the virus entersthe host cell by macropinocytosis [11, 12] the GP is cleaved by hostproteases that remove approximately 80% of the mass of the GP1 subunit,including the mucin-like domain and glycan cap [13, 14]. After cleavageof GP in the endosome, the receptor binding sites on GP become exposed,and the GP1 head then is able to bind its receptor, the Niemann-Pick C1(NPC1) protein [13, 15, 16]. Subsequent conformational changes in GPfacilitate fusion between viral and endosomal membranes. Recognition ofNPC1 by a cleaved GP species (hereafter, GP_(CL)), together with one ormore unknown host signals, is proposed to trigger GP refolding and themembrane fusion reaction that is coupled to it. Endosomal GP→GP_(CL)cleavage is a prerequisite for GP-NPC1 binding and therefore essentialfor filovirus entry.

The dense clustering of glycans on the glycan cap and mucin-like domainlikely shield much of the surface of EBOV GP from humoral immunesurveillance, leaving only a few sites on the EBOV GP protein where nAbscould bind without interference by glycans [17]. Most of our knowledgeabout humoral response against filovirus infections has come fromstudies of murine Abs that recognize EBOV GP. From those studies, it hasbecome clear that mouse neutralizing Abs preferentially target peptidesexposed in upper, heavily glycosylated domains or lower areas (the GP1base) where rearrangements occur that drive fusion of viral and hostmembranes [18]. Abs have not been identified that target proteinfeatures of the membrane proximal external region (MPER) subdomain,which likely rearranges during fusion. KZ52, the first reported humanEBOV GP-specific monoclonal antibody (mAb), was obtained from a phagedisplay library that was constructed from bone marrow RNA obtained froma survivor [19]. KZ52 binds a site at the base of the GP and neutralizesEBOV, most likely by blocking GP-GP_(CL) cleavage and/or inhibiting theconformational changes required for fusion of viral and endosomalmembranes [10]. Some murine Abs also have been reported to bind to thebase region of Ebola virus GPs [20, 21].

The most divergent ebolavirus species (EBOV and SUDV) exhibit 56% GPsequence identity. The sequence identity between filovirus GPs ishighest within the receptor binding region (RBR) [23] and GP2,suggesting that shared epitopes may exist within these domains. SeveralmAbs against EBOV GP with protective efficacy in rodents and non-humanprimates (NHPs) have been reported [3, 5-9, 24, 25]. Neutralizingantibodies have also been described for SUDV with efficacy in a recentlydeveloped rodent model [20, 26]. However, these antibodies bind the sameepitope as KZ52, and like KZ52 are viral species-specific and lackcross-neutralizing or cross-protective properties.

SUMMARY OF THE INVENTION

Described herein are a number of mAbs that are capable of neutralizingEbola viruses both in vitro and in vivo. Surprisingly, the disclosedhuman antibodies possess pan-ebolavirus cross-reactivity andcross-neutralizing activity, and are thus capable of binding andneutralizing all known species of the Ebola virus.

According to a first aspect of the present invention, there are providednovel monoclonal antibodies capable of binding to and neutralizing anEbola virus in a patient. In certain embodiments of the presentinvention, said monoclonal antibodies bind to GP proteins fromebolaviruses belonging to at least two different species, therebyneutralizing the infectivity of viral particles or targeting infectedcells for destruction.

According to a second aspect of the invention, there is providedmonoclonal antibodies comprising the following heavy and light chainCDR3 amino acid sequences:

mAb PE-87-heavy CDR3: SEQ ID No. 1; mAb PE-87-light CDR3: SEQ ID No. 2

mAb PE-24-heavy CDR3: SEQ ID No. 3; mAb PE-24-light CDR3: SEQ ID No. 4

mAb PE-47-heavy CDR3: SEQ ID No. 5; mAb PE-47 light CDR3: SEQ ID No. 6

mAb PE-16-heavy CDR3: SEQ ID No. 7; mAb PE-16-light CDR3: SEQ ID No. 8

mAb PE-05-heavy CDR3: SEQ ID No. 9; mAb PE-05-light CDR3: SEQ ID No. 10

In one embodiment, the critical residues in PE-87 and PE-24 heavy chainCDR3 are D95, W99, and Y100C (Kabat numbering).

In another embodiment of the invention, an antibody isolated asdescribed in Methods (below) from the peripheral B cells of a survivorof a filovirus infection, is modified so that the VH and VL regionnucleotide sequences encode modified V region amino acids that conferenhanced binding capabilities to the mAb. There is provided a method ofpreparing a recombinant antibody comprising: providing a nucleotidesequence selected from the group consisting of

PE-24, PE-87, PE-47, PE-16, PE-64 and PE-05 VH and VL nucleotides;

modifying said nucleic acid sequence such that at least one but fewerthan about 30 of the amino acid residues encoded by said nucleic acidsequence has been changed or deleted without disrupting antigen bindingof said peptide; and expressing and recovering said modified nucleotidesequence.

In yet other embodiments, immunoreactive fragments of any of the hereindescribed monoclonal antibodies are prepared using means known in theart, for example, by preparing nested deletions using enzymaticdegradation or convenient restriction enzymes.

It is another aspect of the present invention to provide modifiedvariants of the disclosed mAb sequences, wherein the sequences have beenaffinity matured or otherwise mutated to increase the therapeuticeffectiveness of the mAb.

Thus, it is one embodiment of the present invention to provide acomposition for the treatment of Ebola, the composition comprising: atherapeutically effective combination of a first monoclonal antibody orantigen binding fragment comprising a heavy chain variable regioncomprising an amino acid sequence at least 90% identical to SEQ. ID NO:12, and affinity matured variants thereof; and a light chain variableregion comprising an amino acid sequence at least 90% identical to SEQID NO: 14, and affinity matured variants thereof; and a pharmaceuticallyacceptable excipient or carrier.

It is another embodiment of the present invention to provide such acomposition, wherein said first monoclonal antibody is binds at leasttwo species of the Flivovirus glycoprotein.

It is yet another embodiment of the present invention to provide such acomposition, wherein the first monoclonal antibody or antigen bindingfragment comprises predominantly a single glycoform.

It is still another embodiment of the present invention to provide sucha composition, wherein the predominantly single glycoform is one ofGnGn, G1/G2, and NaNa.

It is yet another embodiment of the present invention to provide such acomposition, wherein the predominantly single glycoform substantiallylacks at least one of fucose and xylose.

It is second embodiment of the present invention to provide acomposition for the treatment of Ebola, the composition comprising: atherapeutically effective combination of a first monoclonal antibody orantigen binding fragment selected from a list consisting of:

-   -   a. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 15, and affinity matured        variants thereof, and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 18,        and affinity matured variants thereof;    -   b. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 21, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 23,        and affinity matured variants thereof;    -   c. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 29, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 900 identical to SEQ ID NO: 31,        and affinity matured variants thereof;    -   d. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 33, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 900 identical to SEQ ID NO: 35,        and affinity matured variants thereof;    -   e. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 11, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 900/o identical to SEQ ID NO:        13, and affinity matured variants thereof; and a        pharmaceutically acceptable excipient or carrier; wherein said        first monoclonal antibody or antigen binding fragment binds at        least two species of the Flivovirus glycoprotein.

It is another embodiment of the present invention to provide such acomposition, wherein the first monoclonal antibody or antigen bindingfragment comprises predominantly a single glycoform.

It is yet another embodiment of the present invention to provide such acomposition, wherein the predominantly single glycoform is one of GnGn,G1/G2, and NaNa.

It is still another embodiment of the present invention to provide sucha composition, wherein the predominantly single glycoform substantiallylacks at least one of fucose and xylose.

It is third embodiment of the present invention to provide a compositionfor the treatment of Ebola, the composition comprising: atherapeutically effective combination of a first monoclonal antibody orantigen binding fragment is selected from a list consisting of:

-   -   a. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 12, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 14,        and affinity matured variants thereof;    -   b. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 15, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 18,        and affinity matured variants thereof;    -   c. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 21, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 23,        and affinity matured variants thereof;    -   d. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 29, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 31,        and affinity matured variants thereof;    -   e. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 33, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 35,        and affinity matured variants thereof;    -   f. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 11, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 13,        and affinity matured variants thereof; a second monoclonal        antibody or antigen binding fragment, wherein said second        monoclonal antibody or antigen binding fragment binds the Ebola        glycoprotein; and a pharmaceutically acceptable excipient or        carrier.

It is another embodiment of the present invention to provide such acomposition, wherein at least one of the first monoclonal antibody orantigen binding fragment and the second antibody or antigen bindingfragment comprises predominantly a single glycoform.

It is yet another embodiment of the present invention to provide such acomposition, wherein the predominantly single glycoform is one of GnGn,G1/G2, and NaNa.

It is still another embodiment of the present invention to provide sucha composition, wherein the predominantly single glycoform substantiallylacks at least one of fucose and xylose.

It is yet another embodiment of the present invention to provide such acomposition, wherein said therapeutically effective combination furthercomprises a third monoclonal antibody or antigen binding fragment thatbinds to the Ebola glycoprotein.

It is still another embodiment of the present invention to provide sucha composition, wherein said first monoclonal antibody or antigen bindingfragment comprises a heavy chain variable region comprising an aminoacid sequence at least 90%, identical to SEQ. ID NO: 12, and affinitymatured variants thereof; and a light chain variable region comprisingan amino acid sequence at least 90% identical to SEQ ID NO: 14, andaffinity matured variants thereof; and wherein said second monoclonalantibody or antigen binding fragment comprises a heavy chain variableregion comprising an amino acid sequence at least 90% identical to SEQ.ID NO: 15, and affinity matured variants thereof; and a light chainvariable region comprising an amino acid sequence at least 90% identicalto SEQ ID NO: 18, and affinity matured variants thereof.

It is yet another embodiment of the present invention to provide such acomposition wherein said therapeutically effective combination furthercomprises a third monoclonal antibody or antigen binding fragment,wherein said third antibody or antigen binding fragment comprises aheavy chain variable region comprising an amino acid sequence at least90% identical to SEQ. ID NO: 21, and affinity matured variants thereof;and a light chain variable region comprising an amino acid sequence atleast 90% identical to SEQ ID NO: 23, and affinity matured variantsthereof.

It is fourth embodiment of the present invention to provide a method fortreating at least one species of flivovirus infection in a patient, themethod comprising: identifying a patient in need of treatment; andadministering to the patient a therapeutically effective amount of acomposition comprising a combination of: a first monoclonal antibody orantigen binding fragment, wherein said first monoclonal antibody orantigen binding fragment is selected from a list consisting of:

-   -   i. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 12, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 14,        and affinity matured variants thereof;    -   ii. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 15, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 18,        and affinity matured variants thereof;    -   iii. a monoclonal antibody or antigen binding fragment        comprising a heavy chain variable region comprising an amino        acid sequence at least 90% identical to SEQ. ID NO: 21, and        affinity matured variants thereof; and a light chain variable        region comprising an amino acid sequence at least 90% identical        to SEQ ID NO: 23, and affinity matured variants thereof;    -   iv. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 29, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 31,        and affinity matured variants thereof;    -   v. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 33, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 35,        and affinity matured variants thereof;    -   vi. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region comprising an amino acid sequence        at least 90% identical to SEQ. ID NO: 11, and affinity matured        variants thereof; and a light chain variable region comprising        an amino acid sequence at least 90% identical to SEQ ID NO: 13,        and affinity matured variants thereof; and a pharmaceutically        acceptable excipient or carrier.

It is another embodiment of the present invention to provide such amethod, wherein the patient is a mammal.

It is yet another embodiment of the present invention to provide such amethod, wherein the first monoclonal antibody or antigen bindingfragment comprises predominantly a single glycoform.

It is still another embodiment of the present invention to provide sucha method, wherein the predominantly single glycoform substantially lacksat least one of fucose and xylose.

It is fifth embodiment of the present invention to provide a compositionfor the treatment of Ebola, the composition comprising: atherapeutically effective combination of a first monoclonal antibody orantigen binding fragment is selected from a list consisting of:

-   -   a. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region at least 90% identical to a heavy        chain variable region comprising a CDR1 comprising the amino        acid sequence as set forth in SEQ. ID NO: 53, a CDR2 comprising        the amino acid sequence as set forth in SEQ. ID NO: 54, and a        CDR3 comprising the amino acid sequence as set forth in SEQ. ID        NO: 55, and affinity matured variants thereof, and a light chain        variable region at least 90% identical to a light chain variable        region comprising a CDR1 comprising the amino acid sequence as        set forth in SEQ. ID NO: 56, a CDR2 comprising the amino acid        sequence as set forth in SEQ. ID NO: 57, and a CDR3 comprising        the amino acid sequence as set forth in SEQ. ID NO: 58, and        affinity matured variants thereof;    -   b. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region at least 90% identical to a heavy        chain variable region comprising a CDR1 comprising the amino        acid sequence as set forth in SEQ. ID NO: 41, a CDR2 comprising        the amino acid sequence as set forth in SEQ. ID NO: 42, and a        CDR3 comprising the amino acid sequence as set forth in SEQ. ID        NO: 43, and affinity matured variants thereof; and a light chain        variable region at least 90% identical to a light chain variable        region comprising a CDR1 comprising the amino acid sequence as        set forth in SEQ. ID NO: 44, a CDR2 comprising the amino acid        sequence as set forth in SEQ. ID NO: 45, and a CDR3 comprising        the amino acid sequence as set forth in SEQ. ID NO: 46, and        affinity matured variants thereof;    -   c. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region at least 90% identical to a heavy        chain variable region comprising a CDR1 comprising the amino        acid sequence as set forth in SEQ. ID NO: 47, a CDR2 comprising        the amino acid sequence as set forth in SEQ. ID NO: 48, and a        CDR3 comprising the amino acid sequence as set forth in SEQ. ID        NO: 49, and affinity matured variants thereof; and a light chain        variable region at least 90% identical to a light chain variable        region comprising a CDR1 comprising the amino acid sequence as        set forth in SEQ. ID NO: 50, a CDR2 comprising the amino acid        sequence as set forth in SEQ. ID NO: 51, and a CDR3 comprising        the amino acid sequence as set forth in SEQ. ID NO: 52, and        affinity matured variants thereof;    -   d. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region at least 90% identical to a heavy        chain variable region comprising a CDR1 comprising the amino        acid sequence as set forth in SEQ. ID NO: 59, a CDR2 comprising        the amino acid sequence as set forth in SEQ. ID NO: 60, and a        CDR3 comprising the amino acid sequence as set forth in SEQ. ID        NO: 61, and affinity matured variants thereof; and a light chain        variable region at least 90% identical to a light chain variable        region comprising a CDR1 comprising the amino acid sequence as        set forth in SEQ. ID NO: 62, a CDR2 comprising the amino acid        sequence as set forth in SEQ. ID NO: 63, and a CDR3 comprising        the amino acid sequence as set forth in SEQ. ID NO: 64, and        affinity matured variants thereof;    -   e. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region at least 90% identical to a heavy        chain variable region comprising a CDR1 comprising the amino        acid sequence as set forth in SEQ. ID NO: 65, a CDR2 comprising        the amino acid sequence as set forth in SEQ. ID NO: 66, and a        CDR3 comprising the amino acid sequence as set forth in SEQ. ID        NO: 67, and affinity matured variants thereof, and a light chain        variable region at least 90% identical to a light chain variable        region comprising a CDR1 comprising the amino acid sequence as        set forth in SEQ. ID NO: 68, a CDR2 comprising the amino acid        sequence as set forth in SEQ. ID NO: 69, and a CDR3 comprising        the amino acid sequence as set forth in SEQ. ID NO: 70, and        affinity matured variants thereof;    -   f. a monoclonal antibody or antigen binding fragment comprising        a heavy chain variable region at least 90% identical to a heavy        chain variable region comprising a CDR1 comprising the amino        acid sequence as set forth in SEQ. ID NO: 71, a CDR2 comprising        the amino acid sequence as set forth in SEQ. ID NO: 72, and a        CDR3 comprising the amino acid sequence as set forth in SEQ. ID        NO: 73, and affinity matured variants thereof; and a light chain        variable region at least 90% identical to a light chain variable        region comprising a CDR1 comprising the amino acid sequence as        set forth in SEQ. ID NO: 74, a CDR2 comprising the amino acid        sequence as set forth in SEQ. ID NO: 75, and a CDR3 comprising        the amino acid sequence as set forth in SEQ. ID NO: 76, and        affinity matured variants thereof; and a pharmaceutically        acceptable excipient or carrier.

It is another embodiment of the present invention to provide such acomposition, further comprising a second monoclonal antibody or antigenbinding fragment, wherein said second monoclonal antibody or antigenbinding fragment binds the Ebola glycoprotein.

It is yet another embodiment of the present invention to provide such acomposition, wherein said first monoclonal antibody is binds at leasttwo species of the Flivovirus glycoprotein.

It is still another embodiment of the present invention to provide sucha composition, wherein the first monoclonal antibody or antigen bindingfragment comprises predominantly a single glycoform.

It is yet another embodiment of the present invention to provide such acomposition, wherein the predominantly single glycoform is one of GnGn,G1/G2, and NaNa.

It is still another embodiment of the present invention to provide sucha composition, wherein the predominantly single glycoform substantiallylacks at least one of fucose and xylose.

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

DESCRIPTION OF THE FIGURES AND TABLES

Table 1. Amino acid residues comprising CDRs of anti-Ebola mAbs.

FIG. 1 shows a neutralization curve for affinity matured variants of oneembodiment of the present invention.

FIG. 2 shows the binding sites on the EBOV-GP of various embodiments ofthe monoclonal antibodies of the present invention.

FIG. 3 shows the location of the mutations that result in escape mutantresistance to two monoclonal antibodies of the present invention.

FIG. 4 shows neutralization assays preformed against the escape mutants.

FIG. 5 shows survival data for ebolavirus infected guinea pigs treatedwith certain embodiments of the present invention.

FIG. 6 shows immune system response data from ebolavirus infected guineapigs treated with certain embodiments of the present invention.

FIG. 7 shows survival data for ebolavirus infected guinea pigs treatedwith certain embodiments of the present invention.

FIG. 8 shows survival data for ebolavirus infected guinea pigs treatedwith certain embodiments of the present invention.

FIG. 9 survival data for ebolavirus infected guinea pigs treated withcertain embodiments of the present invention.

FIG. 10 survival data for ebolavirus infected non-human primates treatedwith certain embodiments of the present invention.

FIG. 11 survival data for ebolavirus infected non-human primates treatedwith certain embodiments of the present invention.

FIG. 12 survival data for ebolavirus infected non-human primates treatedwith certain embodiments of the present invention.

FIG. 13 shows a neutralization curves for certain embodiments of thepresent invention created using differing production methods.

Table 2 shows the efficiency of anti-GP antibody isolation fromperipheral B cells.

Table 3 shows the cross-reactivity of candidate pan-ebolavirus mAbsagainst different ebolavirus species. Reactivity was measured by ELISA.

Table 4 shows the in vitro neutralization activity and affinities ofcandidate pan-ebolavirus mAbs.

Table 5 shows that mice infected with EBOV and subsequently treated withthe monoclonal antibodies described above showed increased survivalcompared to mice treated with PBS.

Table 6 is a summary of rVSV-GP neutralization by cross-neutralizinghuman mAbs.

Table 7 is a summary of authentic ebolavirus neutralization bycross-neutralizing human mAbs.

Table 8 shows K_(D) values for recognition of EBOV GPΔTM by mature PE-87bearing the indicated mutations in the CDR-H3 loop were determined byBLI. 95% confidence intervals are reported for each binding constant.IC₅₀ values for neutralization of rVSVs bearing ebolavirus GPs by maturePE-87 bearing the indicated mutations in the CDR-H3 loop.

Table 9 shows the mAb protection of mice after challenge with EBOV orSUDV.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned above andhereunder are incorporated herein by reference.

Definitions

As used herein, “neutralizing antibody” (NAb) refers to an antibody, forexample, a monoclonal antibody, capable of disrupting a formed viralparticle or inhibiting formation of a viral particle or prevention ofbinding to or infection of mammalian cells by a viral particle. As usedherein, “diagnostic antibody” or “detection antibody” or “detectingantibody” refers to an antibody, for example, a monoclonal antibody,capable of detecting the presence of an antigenic target within asample. As will be appreciated by one of skill in the art, suchdiagnostic antibodies preferably have high specificity for theirantigenic target. As used herein, “human antibodies” refer to antibodiesthat were isolated from the B cells of a human or directly from thesequence of serum antibodies.

A “therapeutically effective” treatment refers a treatment that iscapable of producing a desired effect. Such effects include, but are notlimited to, enhanced survival, reduction in presence or severity ofsymptoms, reduced time to recovery, and prevention of initial infection.“Therapeutically effective” permutations of a mAb may enhance any of theabove characteristics in a manner that is detectable by routine analysisof patient data. In certain embodiments, such therapeutically effectivemutations include mutations that improve the stability, solubility, orproduction of the mAb, including mutations to the framework regions ofthe mAb sequence.

As used herein, ‘immunoreactive fragment’ refers in this context to anantibody fragment reduced in length compared to the wild-type or parentantibody which retains an acceptable degree or percentage of bindingactivity to the target antigen. As will be appreciated by one of skillin the art, what is an acceptable degree will depend on the intendeduse.

As used herein, a mAb has “pan-Ebola” binding characteristics if it iscapable of binding to at least 2, but preferable more, ebolavirusspecies.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as kappa and lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withineach isotype, there may be subtypes, such as IgG₁, IgG₂, IgG₃, IgG₄,etc. Within light and heavy chains, the variable and constant regionsare joined by a “J” region of about 12 or more amino acids, with theheavy chain also including a “D” region of about 3 or more amino acids.The particular identity of constant region, the isotype, or subtype doesnot impact the present invention. The variable regions of eachlight/heavy chain pair form the antibody binding site.

Thus, an intact antibody has two binding sites. The chains all exhibitthe same general structure of relatively conserved framework regions(FR) joined by three hypervariable regions, also called complementaritydetermining regions or CDRs. The CDRs from the two chains of each pairare aligned by the framework regions, enabling binding to a specificepitope. From N-terminal to C-terminal, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with wellknown conventions [Kabat “Sequences of Proteins of ImmunologicalInterest” National Institutes of Health, Bethesda, Md._(s) 1987 and1991; Chothia, et al., J. Mol. Biol. 196:901-917 (1987); Chothia, etal., Nature 342:878-883 (1989)].

In another embodiment of the invention, there are providedglycoengineered variants of the monoclonal antibodies that containpredominantly a single glycoform. These glycans can be GnGn(GIcNAc₂-Man₃-GlcNAc₂), mono- or di-galactosylated(Gal_((1/2))-GlcNAc₂-Man₃-GlcNAc₂) (hereinaftermono-galactosylated=“G1”, di-galactosylated=“G2”, and a combination ofthe two, in any proportion=“G1/G2”), mono- or di-sialylated(NaNa_((1,2))-Gal_((1/2))-GIcNAc₂-Man₃-GIcNAc₂) containing little or nofucose or xylose. A predominantly single glycoform is any glycoform thatrepresents more than half (e.g. greater than 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%) of all glycoforms present in the antibodysolution.

The RAMP system has been used for glycoengineering of antibodies,antibody fragments, idiotype vaccines, enzymes, and cytokines. Dozens ofantibodies have been produced in the RAMP system by Mapp (5, 6) andothers (7, 8). These have predominantly been IgGs but other isotypes,including IgM (9, 10), have been glycoengineered. Glycoengineering hasalso been extended to human enzymes in the RAMP system (11, 12). Sincethe RAMP system has a rapid turn-around time from Agrobacteriuminfection to harvest and purification (13) patient specific idiotypevaccines have been used in clinical trials for non-Hodgkins lymphoma(7).

For glycoengineering, recombinant Agrobacterium containing a mAb cDNA isused for infection of N. benthamiana in combination with the appropriateglycosylation Agrobacteria to produce the desired glycan profile. Forwild-type glycans (i.e. native, plant-produced glycosylation) wild-typeN. benthamiana is inoculated with only the Agrobacterium containing theanti-M2e cDNA. For the GnGn glycan, the same Agrobacterium is used toinoculate plants that contain little or no fucosyl or xylosyltransfrases (ΔXF plants). For galactosylated glycans, ΔXF plants areinoculated with the Agrobacterium containing the mAb cDNA as well as anAgrobacterium containing the cDNA for β-1,4-galactosyltransferaseexpression contained on a binary Agrobacterium vector to avoidrecombination with the TMV and PVX vectors (14). For sialylated glycans,six additional genes are introduced in binary vectors to reconstitutethe mammalian sialic acid biosynthetic pathway. The genes areUDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase,N-acetylneuraminic acid phosphate synthase, CMP-N-acetylneuraminic acidsynthetase, CMP-NeuAc transporter, β-1,4-galactosylatransferase, andα2,6-sialyltransferase (14).

Glycanalysis of glycoengineered mAbs involved release of N-linkedglycans by digestion with N-glycosidase F (PNGase F), and subsequentderivatization of the free glycan is achieved with anthranilic acid(2-AA). The 2-AA-derivatized oligosaccharide is separated from anyexcess reagent via normal-phase HPLC. The column is calibrated with2-AA-labeled glucose homopolymers and glycan standards. The test samplesand 2-AA-labeled glycan standards are detected fluorometrically.Glycoforms are assigned either by comparing their glucose unit (GU)values with those of the 2-AA-labeled glycan standards or by comparingwith the theoretical GU values (15). Confirmation of glycan structurewas accomplished with LC/MS.

While the RAMP system is an effective method of producing variousglycoengineered and wild-type mABs, it will be recognized that otherexpression systems may be used to accomplish the same result. Forexample, mammalian cell lines (such as CHO or NSO cells [Davies, J.,Jiang, L., Pan, L. Z., LaBarre, M. J., Anderson, D., and Reff, M. 2001.Expression of GnTIII in a recombinant anti-CD20 CHO production cellline: Expression of antibodies with altered glycoforms leads to anincrease in ADCC through higher affinity for FCyRIII. Biotechnol Bioeng74:288-294]), yeast cells (such as Pichia pastoris [Gerngross T.Production of complex human glycoproteins in yeast. Adv Exp Med Biol.2005; 564]) and bacterial cells (such as E. Coli) have been used producesuch mABs.

Described herein are mAbs, designated PE-24, PE-87, PE-47, PE-16, PE-64and PE-05, which have surprisingly exhibited pan-Ebola neutralizingcharacteristics. The preferred antibodies of the present inventioncomprise mAbs with amino acid sequences sufficiently identical toreferenced amino acid sequences. By “sufficiently identical” is intendedan amino acid sequence that has at least about 60% or 65% sequenceidentity, about 70% or 75% sequence identity, about 80% or 85% sequenceidentity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to a reference sequence using one ofthe alignment programs known in the art.

The sequences below show the amino acid modifications to mAb PE-64 VHand VL amino acids to yield mAb PE-47 (Modifications are shown in Bold,CDR sequences are Underlined).

mAb PE-64 VH amino acids: SEQ ID No. 11EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTIDYAAPVKGRFTISRDDSKNTVYLQMTSLKTEDTAVYYCTTYTEDMRYFDWLLRGGETFDYWGQGTLVTVSS mAb PE-47 VH amino acids: SEQ ID No. 12EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGEGLEWVGRIKSKTDGGTIDYAAPVKGRFTISRDDSKNTVYLQMTSLKTEDTAVYYCTTYTEDMQYFDWLLRGGETFDYWGQGTLVTVSS mAb PE-64 VL amino acids: SEQ ID No. 13DIRLTQSPSSLSASVGDRVTITCRASHYISTYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGFGTDFSLTISSLQPEDFATYHCQQSYSTPGRYTF GQGTKVEIKmAb PE-47 VL amino acids: SEQ ID No. 14DIQMTQSPSSLSASVGDRVTITCRASQYISTYLNWYQQKPGKAPKLLIYAAYNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPGRYTF GQGTKVEIK

The antibodies displayed below were isolated from the peripheral B cellsof a survivor of the 2014 Ebola virus outbreak in West Africa (CDR aminoacids are disclosed in Table 1).

PE-87 VH amino acids: SEQ ID No. 15

PE-87 VH nucleotides: SEQ ID No. 16

An alternative PE-87 VH amino acid sequence is: SEQ ID No. 17(alterations shown in Bold and Underlined)

E VQLV E SG GG LVQPGGSLRVSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGLGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDHRVWAAGYHFDYWGQG TLVTVSS

PE-87 VL amino acids: SEQ ID No. 18

PE-87 VL nucleotides: SEQ ID No. 19

An alternative PE-87 VL amino acid sequence is: SEQ ID No. 20(alterations shown in Bold and Underlined)

DI QM TQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGEAPKLLISDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYSSPTFGGG TKVEIK

PE-24 VH amino acids: SEQ ID No. 21

PE-24 VH nucleotides: SEQ ID No. 22

PE-24 VL amino acids: SEQ ID No. 23

PE-24 VL nucleotides: SEQ ID No. 24

PE-47 VH amino acids: SEQ ID No. 25

PE-47 VH nucleotides: SEQ ID No. 26

PE-47 VL amino acids: SEQ ID No. 27

PE-47 VL nucleotides: SEQ ID No. 28

PE-16 VH amino acids: SEQ ID No. 29

PE-16 VH nucleotides: SEQ ID No. 30

PE-16 VL amino acids: SEQ ID No. 31

PE-16 VL nucleotides: SEQ ID No. 32

PE-05 VH amino acids: SEQ ID No. 33

PE-05 VH nucleotides: SEQ ID No. 34

PE-05 VL amino acids: SEQ ID No. 35

PE-05 VL nucleotides: SEQ ID No. 36

PE-64 VH amino acids: SEQ ID No. 37

PE-64 VH nucleotides: SEQ ID No. 38

PE-64 VL amino acids: SEQ ID No. 39

PE-64 VL nucleotides: SEQ ID No. 40

TABLE 1 Amino acid residues comprising CDRs of anti-Ebola mAbs(SEQ ID Nos. indicated in parenthesis) Mab V region CDR 1 CDR 2 CDR 3PE-87 VH GFTFSSYAMS (41) AISGLGGSTYYADSV (42) DHRVWAAGYHFDY (43)PE-87 VL RASQSISSWLA (44) DASSLES (45) QQYYSSPT (46) PE-24VHGFTFSSYAMS (47) EISGLGGSTYYADSAK (48) DHRVWAPGYYFDH (49) PE-24 VLRASQSISSWLA (50) DASSLES (51) QQYNRSPT (52) PE-47 VH GFTFSNAWMS (53)RIKSKTDGGTIDYAAPVK (54) YTEDMQYFDWLLRGGETFDY (55) PE-47 VLRASQYISTYLN (56) AAYNLQS (57) QQSYSTPGRYT (58) PE-16 VH GYTFTTYYMH (59)IINPSGGITRYAQKFQ (60) DRYPVLFATDYGMDV (61) PE-16 VL RASQSVSGYLA (62)DASNRAT (63) QQRSIWPPGVT (64) PE-05 VH GFTFGDYAMS (65)FLRSKAYGGTAEYAASVK (66) DGFRGSSWGYSYYGMDV (67) PE-05 VLSGSSSNIGGNTVS (68) TNDQRPS (69) WDDSLNGPVFGGGT (70) PE-64 VHGFTFSNAWMS (71) RIKSKTDGGTIDYAAPVK (72) YTEDMRYFDWLLRGGETFDY (73)PE-64 VL RASHYISTYLN (74) AASNLQS (75) QQSYSTPGRYT (76)

In certain embodiments of the present invention, the above mAb sequencesare affinity matured to enhance binding or otherwise improve thetherapeutic efficacy of the antibody. In one embodiment, optimization ofantibodies was performed via a light chain diversification protocol, andthen by introducing diversities into the heavy chain and light chainvariable regions as described below:

CDRL1 and CDRL2 selection: The CDRL3 of a single antibody was recombinedinto a premade library with CDRL1 and CDRL2 variants of a diversity of1×10⁸ and selections were performed with one round of MACS and fourrounds of FACS. For each FACS round the libraries were affinitypressured using titrating amounts of an ebolavirus GP (for example, SUDVGP) and sorting was performed in order to obtain a population with thedesired characteristics.

VH Mut selection: The heavy chain variable region (VH) was mutagenizedvia error prone PCR. The library was then created by transforming thismutagenized VH and the heavy chain expression vector into yeast alreadycontaining the light chain plasmid of the parent. Selections wereperformed similar to previous cycles using FACS sorting for two rounds.For each FACS round the libraries were affinity pressured usingtitrating amounts of Sudan GP and sorting was performed in order toobtain a population with the desired characteristics.

ADI-23774 (PE-47) was generated by combining the most improved HC (fromthe VH mut selection) with the most improved LC (from the L1/L2selection).

FIG. 1 illustrates the enhanced neutralization potential of the parent(PE-64), best VH mutant, best VL mutant, and best VH/VL mutant (PE-47).

It will be apparent to those having skill in the art that these oralternate methods of affinity maturation may be used to rapidly andefficiently improve upon the desired characteristics of the mAbsequences described herein, and that routine analytical tools may beused to identify if any potential variant developed using thesetechniques possess the desired characteristic.

These antibodies have high affinity and avidity for Ebola glycoproteins,which means that in certain embodiments they can be used as therapeuticreagents administered to an individual with an ebolavirus infection oras prophylactic reagents to prevent an ebolavirus infection or as highlysensitive diagnostic tools. In particular, we have found that PE-87 andPE-47 act primarily at a step that follows GP→GP_(CL) cleavage andreceptor engagement. Endosomally generated GP_(CL) species (either aloneor in complex with NPC1) is the presumptive final target of these mAbs.Strikingly, GP cleavage to GP_(CL) enhanced the antiviral potencies ofPE-64, PE-87, and PE-47 by 50-200 fold. Together, these results suggestthat the broadly neutralizing mAbs PE-87 and PE-47 differ frompreviously described monospecific mAbs (KZ52, c2G4, and 4G7), in theirability to target and neutralize a cleaved GP species that is generateddeep in the endocytic pathway. Conversely, the latter mAbs appear to actprincipally at and/or prior to the GP→GP_(CL) cleavage step. PE-64displayed a dual behavior, and may act both upstream, to block GPcleavage, and downstream, to target one or more GP_(CL)-like species ator near the membrane fusion step. We assessed the protective efficacy ofthese broadly neutralizing human mAbs in three small-animal models oflethal ebolavirus challenge. First, wild type (WT) BALB/c mice wereexposed to mouse-adapted EBOV (EBOV-MA), and then administered a singledose of each mAb at 2 days post-infection (300 μg/animal).Cross-neutralizing mAbs were highly (≥80%) protective against EBOV inthis stringent post-exposure setting, with little or no weight lossapparent in mAb-treated animals.

FIG. 2 illustrates negative stain EM reconstructions of broadlyneutralizing ebolavirus mAbs. A structure of ebolavirus GP (based on PDBIDs:5JQ3) displaying the antigenic surfaces and corresponding structuralregions of interest. The disordered mucin domain (dashed lines), GP1,GP2, fusion loop, glycan cap in, CHR2 region and the N563-linked glycan.Top and side views are shown for negative stain EM 3D reconstructions ofFab models of PE-87, PE-47, PE-24 and PE-16 (shown in dark gray) incomplex with EBOV GP.

We next evaluated the NAbs in the Type I interferon α/βreceptor-deficient mouse model for SUDV challenge. Mice were exposed toWT SUDV, and then dosed with each NAb on days 1 and 3 post-infection(300 μg/animal/dose). The pan-ebolavirus mAbs PE-87 and PE-47 afforded≥95% survival and greatly reduced weight loss, relative to the PBScontrol group. By contrast, PE-16 and PE-64, both weak SUDVneutralizers, provided little or no protection against SUDV.

Finally, we tested the anti-BDBV efficacy of the two pan-ebolavirushuman mAbs, PE-87 and PE-47, in the domestic ferret, which is the onlydescribed non-NHP model for BDBV challenge. Animals received two dosesof each NAb (15 mg and 10 mg per animal on days 3 and 6 post-challenge,respectively). As observed previously, BDBV infection was uniformlylethal, with PBS-treated animals succumbing between days 8-10 followingchallenge. By contrast, both mAbs afforded highly significant levels ofsurvival (3 of 4 animals for PE-87; 2 of 4 for PE-24). Furthermore, peakviremia levels correlated with mAb treatment and survival outcome, withlower viral titers observed in the surviving animals relative to thosethat succumbed to infection (p<0.001), and in mAb-treated animalsrelative to PBS-treated controls (p<0.001). Viremia also trended lowerin animals receiving PE-87 relative to those receiving PE-47, but thisdifference did not reach statistical significance. In sum, our findingsdemonstrate that the pan-ebolavirus mAbs PE-87 and PE-47 can affordpost-exposure protection against challenge by the three divergentebolaviruses currently associated with lethal disease outbreaks inhumans.

In another embodiment of the present invention, the mAbs of the presentinvention have been shown to provide complete protection to a non-humanprimate model of Ebola virus challenge. Four days after exposure to alethal challenge of EBOV virus, a group of rhesus macaque monkeys weretreated with either one dose of an NAb cocktail (comprising 25 mg/kgeach of PE-87 and PE-47) or two doses of the same NAb cocktail (one at 4days post infection, comprising 50 mg/kg of the NAbs, and another at 7days post infection, comprising 25 mg/kg of the NAbs). As previouslyobserved, EBOV infection was uniformly lethal, with the all PBS-treatedanimals succumbing by the 7^(th) day post infection. By contrast, everyanimal from the NAb treatment groups survived, with no detectable viralRNA present in the blood of the treatment groups 10 days following theinitial treatment, as assayed via qRT-PCR.

The NAb cocktail of PE-87 and PE-47 (also referred to herein as MBP134)was further tested as follows. First, escape mutants that were resistantto the individual components of MBP134 were generated. Escape mutantselections were performed by serial passage of rVSV-GP particles in thepresence of test antibody. Briefly, serial 3-fold dilutions of viruswere preincubated for one hour with a concentration of antibodycorresponding to the IC₉₀ value derived from neutralization assays, andthen added to confluent monolayers of Vero cells in 12-well plates, induplicate. Infection was allowed to proceed to completion (>90% celldeath by eye), and supernatants were harvested from the infected wellsthat received the highest dilution (i.e., the least amount) of viralinoculum. Following three subsequent passages under antibody selectionwith virus-containing supernatants as above, supernatants from passage 4were tested for viral neutralization escape. If resistance was evident,individual viral clones were plaque-purified on Vero cells, and their GPgene sequences were determined as described previously (Wong et al.,2010).

FIG. 3 illustrates the mutations to the rVSV-GP and their relativelocations within the three-dimensional structure of the viralglycoprotein for the two escape mutants that were most resistant toPE-47 (MBPO47) and PE-87 (MBP087) respectively. Namely, the PE-87 escapemutant contained a G528E substitution, while the PE-47 escape mutantcontained a N514D substitution.

FIG. 4 illustrates the dose response curves of the above-mentionedescape mutants and the wild-type SUDV virus to concentrations PE-47 andPE-87. Importantly with regard to the efficacy of a multi-mAb cocktail,the escape mutations which provided resistance to one mAb resulted insignificantly enhanced neurtralization by the other. As such, in certainembodiments of the present invention, a combination of multipleantibodies is provided which significantly reduce the risk of viralresistance development.

As noted above, antibodies comprising a substantially single glycan andlacking fucose show enhanced efficacy in patients. To determine ifafucosylated MBP134 has increased efficacy in mammals, fucosylated andafucosylated versions of the cocktail were used to treat guinea pigschallenged with a lethal dose of EBOV. All guinea pigs were healthy andimmune competent as per vendor's representation. All guinea pigs weredrug and test naive. Animals were monitored daily for food and waterconsumption and given environmental enrichment according to theguidelines for the species. Cleaning of the animals was completed threetimes per week which included a complete cage and bedding materialchange. Animals were kept two or three per cage in the large shoe boxcages from IVC Alternative Design. Each unit is ventilated with a HEPAblower system. 4-6 week old female Hartley guinea pigs (250-300 g) wererandomly assigned to experimental groups and challenged via IP with a1000×LD50 of guinea pig adapted EBOV/Mayinga in 1 mL of DMEM. EitherMBP134 or the afucosylated MBP134-N was given IP at indicated timepoints and doses, with 6 guinea pigs/group (n=6). Control guinea pigswith 4 animals/group (n=4), were given PBS treatment. Animals wereobserved for clinical signs of disease, survival and weight change for15-16 days, while survival was monitored for an additional 12 days.

FIG. 5 illustrates the survival curves of the afucosylated vs.fucosylated MBP134 at various doses. The afucosylated cocktail showeddramatically improved survival, even at the lowest dosage tested.Furthermore, blood drawn from the animals showed significantly increasedimmune reactions in response to treatment with afucosylated PE-47 andPE-87, as compared to their fucosylated counterparts and other anti-EBOVmAbs c13C6 (also afucosylated) and 2G12, as illustrated in FIG. 6. Thus,in certain embodiments of the present invention, there is provided amonoclonal antibody that substantially lacks fucose.

To determine the ability of the afucoslyated MBP134 to neutralizemultiple strains of the ebolavirus, a dose down study of guinea pigsinfected with a lethal dose of SUDV was conducted. As illustrated inFIG. 7, animals treated at three and four days post infection had 100%survival, while even treatment at 5 dpi resulted in a dramatic increasein survival. To determine if a lower dose of MBP134 would be effectiveat 4 dpi, and if a higher dose would lead to increased survival ifadministered at 5 dpi, further tests were conducted. As illustrated inFIG. 8, reduced doses of MBP134 administered at 4 dpi resulted inexcellent, though not perfect, survival rates among the treated animals.Furthermore, doubling the dose administered at 5 dpi resulted in all ofthe infected animals surviving. In certain embodiments of the presentinvention, the increase dosage of the monoclonal antibodies at laterdates post infection allows the host animals to overcome the increasedviral load associated with the infection.

Thus, in certain embodiments of the present invention, a patient istreated with an effective dose of a monoclonal antibody or combinationof monoclonal antibodies. An effective dose includes, but is not limitedto, 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, and 100mg/kg

To further explore the ability of the monoclonal antibodies disclosedherein to protect against multiple strains of the ebolavirus in mammals,female ferrets were infected with various strains of ebolavirus andtreated with different dosages of MBP134. Female ferrets weighing 0.75-1kg were housed 2-3 per cage per study. Ferrets were anesthetized byintramuscular injection with a ketamine-acepromazine-xylazine cocktailprior to all procedures. Prior to challenge, transponder chips(Bio-Medic Data Systems) were subcutaneously implanted foridentification and temperature monitoring. Subjects were challengedintranasally with a lethal dose of 1000 plaque-forming units (PFU) ofZEBOV strain Kikwit, SEBOV strain Gulu, or BDBV and treated withMBP134-N at the times and dosing shown in FIG. 9. As shown in FIG. 9,two doses of 15 mg at two or three dpi and five or six dpi weresufficient to offer full survival to the infected mammals. Furthermore,the results illustrated here, combined with those discussed above,indicate that the MBP134 cocktail provides protection against manydifferent stains of ebolavirus in mammals.

To determine if this protection extends to primates, rhesus macaqueswere infected with a lethal dose of EBOV/Kikwit and treated with themonoclonal antibodies of the present invention. Rhesus macaques at UTMBwere challenged by intramuscular injection (IM) with 1,000 PFU ofEBOV/Kikwit. Two treatment groups (n=4/group) were treated either with asingle 25 mg/kg dose of MBP134-N on day 4 or two doses of MBP134-N day 4(50 mg/kg) and day 7 (25 mg/kg) post infection. Control animals (n=2)were treated with PBS. All the macaques were given physical examinationsand blood was collected at the time of viral challenge; and on days 4,7, 10, 14, 21, and 28 after challenge. The macaques were monitored dailyand scored for disease progression with an internal filovirus scoringprotocol approved by the UTMB Institutional Animal Care and UseCommittee (IACUC) in accordance with state and federal statutes andregulations relating to experiments involving animals and by the UTMBInstitutional Biosafety Committee. The scoring changes measured frombaseline included posture/activity level; attitude/behavior; food andwater intake; weight; respiration; and disease manifestations, such asvisible rash, hemorrhage, ecchymosis, or flushed skin, with increasedscores resulting in euthanasia. As illustrated in FIG. 10, all of thetreated primates survived the lethal challenge of ebolavirus.

As illustrated in FIG. 11, the protection offered to primates by theantibodies of the present invention extends to multiple strains ofebolavirus. Even a single dose of MBPl34 is sufficient to protect from alethal challenge of both SUDV/Nza-Boniface and SUDV/Gulu in rhesusmacaques.

Furthermore, the monoclonal antibodies of the present invention provideprotection from ebolavirus challenge in different species of primate.Cynomolgus monkeys at UTMB were challenged by intramuscular injection(IM) with 1,000 PFU of BDBV (200706291 Uganda isolate, Vero E6 passage2). One treatment group (n=6) was treated with a single 25 mg/kg dose ofMBP134 (from CHOK1-AF) on day 7 post infection via IV infusion. Controlanimals (n=3) were untreated. All the animals were given physicalexaminations and blood was collected at the time of viral challenge; andon days 4, 7, 10, 14, 21, and 28 after challenge (or at time ofeuthanasia). All animals were monitored daily and scored for diseaseprogression with an internal filovirus scoring protocol approved by theUTMB Institutional Animal Care and Use Committee. The scoring changesmeasured from baseline included posture/activity level,attitude/behavior, food intake, respiration, and disease manifestationssuch as visible rash, hemorrhage, ecchymosis, or flushed skin. A scoreof >9 indicated that an animal met criteria for euthanasia. Asillustrated in FIG. 12, a single dose of MBP134 as late as one-week postinfection is sufficient to offer excellent protection.

In order to optimize the production methodology of the monoclonalantibodies disclosed herein, the ability of PE-87 and PE-47 produced inplants or CHO cells to neutralize numerous strains of ebolavirus weretested. As illustrated in FIG. 13, monoclonal antibodies produced inboth plant and CHO based systems possess similar neurtralizationcharacteristics. As such, these, or other systems known in the art, maybe used to produce the monoclonal antibodies of the present invention.

It is of note that as discussed herein, any of the above describedantibodies may be formulated into a pharmaceutical treatment forproviding passive immunity for individuals suspected of or at risk ofdeveloping hemorrhagic fever comprising a therapeutically effectiveamount of said antibody. The pharmaceutical preparation may include asuitable excipient or carrier. See, for example, Remington: The Scienceand Practice of Pharmacy, 1995, Gennaro ed. As will be apparent to oneknowledgeable in the art, the total dosage will vary according to theweight, health and circumstances of the individual as well as theefficacy of the antibody. While the preferred embodiments of theinvention have been described above, it will be recognized andunderstood that various modifications may be made therein, and theappended claims are intended to cover all such modifications which mayfall within the spirit and scope of the invention.

Materials and Methods 1 Human Subjects

Human blood samples were collected after Institutional Review Board(IRB) approval of a protocol to isolate B cells from healthy adultvolunteers to identify antibodies elicited from prior immunization orinfections. Eligible subjects were determined based on immunization andinfection history recorded on a self-reported questionnaire completedprior to sample collection. Peripheral blood mononuclear cells wereobtained from a survivor of the 2014 EBOV outbreak three months afterthe patient had been diagnosed with EBOV infection.

B Cell and Plasma Isolation

Approximately 85 ml of whole blood was collected in 8.5 ml ACD SolutionA Vacutainer® venous blood collection tubes (Becton Dickinson) per themanufacturer's protocol. Blood was transported at room temperature anddistributed into 50 ml conical tubes before addition of 300 μl ofRosetteSep™ human B cell enrichment cocktail (StemCell Technologies) per21 ml of blood, mixed by inversion and incubated for 20 minutes at roomtemperature. The total volume was brought to 50 ml with Hank's BalancedSalt Solution (HBSS), layered over Ficoll-Paque Plus (GE Healthcare) andcentrifuged following the manufacturer's protocol. The B cell layer wasremoved from the density gradient by pipette, washed twice in HBSS bycentrifugation at 400×g, frozen at 6.5×106 cells/ml in a 1:1 mixture ofFBS (Life Technologies) and cryoprotective medium (Lonza) and storedunder liquid nitrogen. Plasma was collected from the top layer of thedensity gradient and stored at −80° C. until use.

TABLE 2 Efficiency of anti-GP mAb isolation from peripheral B cells.Total number of IgG+ B cells sorted: 600 Number of antibodies cloned:420 (70%) Number of clones expressing IgG: 378 (63%) Number of EBOV GPbinders: 349 (58%)

Anti-EBOV GP Plasma ELISA

A high-binding ELISA plate was coated with 1 μg/ml of EBOV rGPΔTM (IBTBioSciences) diluted in PBS overnight at 4° C. After washing, wells wereblocked with 1% BSA in PBS and 0.05% Tween-20 for 2 hours at roomtemperature. Wells were washed and serial dilutions of human plasma(diluted in blocking buffer) were added and incubated for 1.5 hours atroom temperature. As positive and negative controls, serial dilutions ofmAb KZ52 (IBT BioSciences) or an irrelevant human mAb, respectively,were added to appropriate wells. After washing, HRP-conjugated donkeyanti-human IgG (Jackson ImmunoResearch) or HRP-conjugated goatanti-human IgA (Southern Biotech) secondary antibody was incubated inappropriate wells for 1.25 hours at room temperature. Wells were washedtwice and developed with SureBlue TMB substrate (KPL). The reaction wasstopped with 1M HCl and wells were read on an EMax Microplate Reader(Molecular Devices) at 450 nm wavelength. Plasma endpoint titers weredetermined by calculating the highest serum dilution that gives areading above the blank including three standard deviations.

Single B Cell Sorting

Purified B cells were stained using anti-human IgM (BV605), IgD (BV605),IgG (BV421), CD8 (APC-Cy7), CD14 (AF700), CD19 (PerCP-Cy5.5), CD20(PerCP-Cy5.5) and biotinylated EBOV GPΔTM. Biotinylated GPΔTM was usedat a concentration of 50 nM and detected using streptavidin-APC (LifeTechnologies) at a dilution of 1:500. Single cells were sorted on aMoFlo cytometer (Beckman-Coulter) into 96-well PCR plates (BioRad)containing 20 μl/well of lysis buffer [5 μl of 5× first strand cDNAbuffer (Invitrogen), 0.5 μl RNaseOUT (Invitrogen), 1.25 μldithiothreitol (Invitrogen), 0.625 μl NP-40 (New England Biolabs), and12.6 μl dH2O]. Plates were immediately frozen on dry ice before storageat −80° C.

Amplification and Cloning of Antibody Variable Genes

Single B cell PCR was performed essentially as previously described[27]. Briefly, IgH, Igλ and Igκ variable gene transcripts were amplifiedby RT-PCR and nested PCR reactions using cocktails of primers specificfor IgG [27]. The primers used in the second round of PCR contained 40base pairs of 5′ and 3′ homology to the cut expression vectors to allowfor cloning by homologous recombination into Saccharomyces cerevisiae[28]. PCR products were cloned into S. cerevisiae using the lithiumacetate method for chemical transformation [29]. Each transformationreaction contained 20 μl of unpurified heavy chain and light chain PCRproduct and 200 ng of cut heavy and light chain plasmids. Individualyeast colonies were picked for sequencing and down-streamcharacterization.

Expression and Purification of Antibodies and Fab Fragments

Antibodies used for binding experiments, competition assays,neutralization assays, and structural studies were expressed inSaccharomyces cerevisiae cultures grown in 24 well plates. After 6 daysof growth, the yeast cell culture supernatant was harvested bycentrifugation and subject to purification. IgGs used in protectionexperiments were expressed by transient co-transfection of heavy andlight chain plasmids into HEK293 cells. One day prior to transfection,HEK293 cells were passaged at 2.0-2.5×106 cells/ml. On the day oftransfection, cells were pelleted by centrifuging at 400 g for 5 min,and cell pellets were resuspended in fresh FreeStyle F17 medium at adensity of 4×106 cells/ml and returned to the incubator. A transfectionmixture was prepared by first diluting the plasmid DNA preparations inFreeStyle F17 medium (1.33 μg total plasmid DNA per ml of culture).Transfection agent, PEIpro™ (Polyplus Transfection, Illkirch, France),was then added to the diluted DNA at a DNA-to-PEI ratio of 1:2, and themixture was incubated at room temperature for 10 min. The transfectionmixture was then added to the culture. Cultures were harvested six dayspost transfection by two rounds of centrifugation, each at 2000×g for 5min, and the clarified conditioned medium subject to antibodypurification. Cell supernatents were purified by passing over Protein Aagarose (MabSelect SuRe™ from GE Healthcare Life Sciences). The boundantibodies were washed with PBS, eluted with 200 mM acetic acid/50 mMNaCl pH 3.5 into ⅛^(th) volume 2M Hepes pH 8.0, and buffer-exchangedinto PBS pH 7.0. Fabs were generated by digesting the IgGs with papainfor 2 h at 30° C. The digestion was terminated by the addition ofiodoacetamide, and the Fab and Fc mixtures were passed over Protein Aagarose to remove Fc fragments and undigested IgG. The flowthrough ofthe Protein A resin was then passed over CaptureSelect™ IgG-CH1 affinityresin (ThermoFischer Scientific), and eluted with 200 mM acetic acid/50mM NaCl pH 3.5 into ⅛th volume 2M Hepes pH 8.0. Fab fragments then werebuffer-exchanged into PBS pH 7.0.

Expression and Purification of EBOV GPs

Recombinant EBOV GP ectodomains containing the mucin-like domain (EBOVGPΔTM) or lacking residues 312-463 of the mucin-like domain (EBOVGPΔmuc) were produced as described previously [10, 30].

EBOV GPΔTM Biotinylation

EBOV GPΔTM was biotinylated using EZ-Link™ Sulfo-NHS-LC-Biotin (LifeTechnologies) followed by a desalting step by a Zeba™ Spin DesaltingColumn (Life Technologies).

Biolayer Interferometry Binding Analysis

IgG binding to the different GP antigens was determined by BLImeasurements using a ForteBio Octet HTX instrument (Pall Life Sciences).For high-throughput KD screening, IgGs were immobilized on AHQ sensors(Pall Life Sciences) and exposed to 100 nM antigen in PBS containing0.1% BSA (PBSF) for an association step, followed by a dissociation stepin PBSF buffer. Data was analyzed using the ForteBio Data AnalysisSoftware 7. The data was fit to a 1:1 binding model to calculate anassociation and dissociation rate, and KD was calculated using the ratiokd/ka.

Anti-GP mAb ELISAs

ELISA plates were coated with 50 μl PBS containing 4 μg/mL EBOV GPantigens for 1 h at room temperature. After washing, wells were blockedwith 3% BSA for 1 h at room temperature. After removal of the blockingsolution, mAbs were applied to the plates at a concentration of 0.2μg/ml and incubated at room temperature for 1 h. After washing, bindingwas detected with an anti-human HRP-conjugated secondary antibody andTMB substrate. Optical density was read at 450 nm.

TABLE 3 Cross-reactivity of pan ebolavirus mAbs (elisa) mAb EBOV SUDVBDBV RESTV TAFV MARV sGP GPcl PE-24 YES YES YES YES YES NO NO YES PE-05YES YES YES YES YES NO YES NO PE-87 YES YES YES YES YES NO NO YES PE-16YES WEAK YES NO YES NO NO YES PE-47 YES YES YES NP YES NO NO YES

Antibody Competition Assays

Antibody competition assays were performed essentially as previouslydescribed [31]. Antibody competition was measured by the ability of acontrol anti-EBOV GP Fab to inhibit binding of yeast surface-expressedanti-GP IgGs to GPΔmuc. 50 nM biotinylated GPΔmuc was pre-incubated with1 μM competitor Fab for 30 min at RT and then added to a suspension ofyeast-expressed anti-GP IgG. Unbound antigen was removed by washing withPBSF. After washing, bound antigen was detected using Streptavidin AlexaFluor 633 at a 1:500 dilution (Life Technologies) and analyzed by flowcytometry using a BD FACS Canto II. Results are expressed as the foldreduction in antigen binding in the presence of competitor Fab relativeto an antigen-only control.

Neutralization Assays

Virus-specific neutralizing antibody responses were titrated essentiallyas previously described [32]. Briefly, plasma or antibodies were dilutedserially in Minimal Essential Medium (Corning Cellgro, Manassas, Va.)containing 5% heat-inactivated fetal bovine serum (Gibco-Invitrogen,Gaithersburg, Md.), 1× Anti-Anti (Gibco-Invitrogen, Gaithersburg, Md.)(MEM complete) and incubated 1 hour at 37° C. with virus. Afterincubation, the antibody-virus or plasma-virus mixture was added induplicate to 6-well plates containing 90-95% confluent monolayers ofVero E6 cells. Plates were incubated for 1 hour at 37° C. with gentlerocking every 15 minutes. Following the incubation, wells were overlaidwith 0.5% agarose in supplemented EBME media, 10% heat-inactivated fetalbovine serum (Gibco-Invitrogen, Gaithersburg, Md.), 2× Anti-Anti(Gibco-Invitrogen, Gaithersburg, Md.), and plates were incubated at 37°C., 5% CO2 for 7 days. On day 7, cells were stained by the addition of asecond overlay prepared as above containing 4-5% neutral red. Plateswere incubated for 18-24 hours at 37° C., 5% CO2. The endpoint titer wasdetermined to be the highest dilution with a 50% or greater or 80% orgreater reduction (PRNT50, PRNT80) in the number of plaques observed incontrol wells. The assay limit of detection was calculated to be 5plaque forming units (p.f.u.)/ml by this method.

TABLE 4 Candidate pan-Ebolavirus mAbs in vitro activity NeutralizationNeut. (VSV-GP IC₅₀, nM) WT EBOV Microneut. WT Affinity EBOV PRNT₅₀ IC₅₀(nM) mAb Epitope (KD, nM) EBOV SUDV BDBV RESTV TAFV GP_(CL) (nM) EBOVSUDV BDBV PE-05 GC 44 8.8 34 3.7 22 0.8 NR 4.0 7.7 NR NR PE-87 IFL <0.010.5 0.3 0.5 0.2 1.0 0.2 <0.05 0.3 0.3 0.4 PE-16 Stalk 0.16 0.2 NR 0.6 NR4.8 5.7 <0.02 0.05 NR 0.2 PE-47 Other 3.5 6.6 5.1 0.4 NR 6.1 0.08 NT 0.7<0.1  0.5 PE-24 IFL 1 1.8 0.5 0.8 0.2 1.5 0.6 0.4 1 0.4 0.3 Epitopeanalyses and affinity measurements were performed by both Mapp andIntegrated BioTherapeutics. VSV-GP assays were performed in Dr. K.Chandran's laboratory (Albert Einstein); neutralization assays withwildtype virus were performed in Dr. J. Dye's lab (USAMRIID); IFL =internal fusion loop; GC = glycan cap; RBS = receptor binding site;GP_(CL) = cleaved GP, the form of GP exposed in the endosome when virusis internalized by the cell in preparation for fusion with the host cellreceptor; P = in progress; NR = non-reactive; NT = not tested; WT =wildtype.

Single-Particle Electron Microscopy

For all EM studies the EBOV GPΔTM construct described above was used.Fabs were generated as described above and incubated with the EBOV GPΔTMtrimer at a ratio of 1:10 for overnight at 4° C. Complexes were thendeposited onto a carbon coated copper mesh grid and stained with 1%uranyl formate. Samples were imaged on a Tecnai F12 microscope using theautomated image acquisition software Leginon [33]. Images were collectedat 52,000× magnification resulting in a final pixel size at the specimenlevel of 2.05 A using a Tietz 4K CMOS detector. Images wereautomatically uploaded to and processed within our Appion database [34].Individual complexes were extracted from raw images using DogPicker [35]binned by 2 and placed into a stack. The stack was then subjected toreference free 2 dimensional classification using MRA/MSA [PMID14572474]. Class averages that did not respond to Fab-EBOVΔTM complexeswere removed from all subsequent analyses. A subset of 2D class averageswas used to create an initial model using common lines within EMAN2[36]. The raw particle stack was then refined against the initial modelusing EMAN2 to yield the final 3D volumes. UCSF Chimera was used formodeling and figure generation [37].

EBOV Challenge Studies in Mice

The lethal mouse-adapted EBOV mouse model was developed at the U.S. ArmyMedical Research Institute of Infectious Diseases (USAMRIID) by serialpassages of EBOV (Zaire) in progressively older suckling mice [38].Female BALB/c mice, aged 6 to 8 weeks, were purchased from Charles RiverLaboratory. Upon arrival, mice were housed in microisolator cages in ananimal biosafety level 4 containment area and provided chow and water adlibitum. On day 0, mice were infected intraperitoneally (i.p.) with 100p.f.u. of mouse-adapted EBOV. Two days post-infection, groups of mice(10 mice per group) were treated i.p. with a single dose (100 μg) ofantibody. Negative control mice received PBS. Mice were monitored daily(twice daily if there were clinical signs of disease) for 28 dayspost-infection. Group weights were taken on days 0-14, and on days 21and 28 post-infection. Survival was compared using the log-rank test inGraphPad PRISM 5. Differences in survival were considered significantwhen the P value was less than 0.05. Research was conducted under anIACUC approved protocol in compliance with the Animal Welfare Act, PHSPolicy, and other Federal statutes and regulations relating to animalsand experiments involving animals. The facility where this research wasconducted is accredited by the Association for Assessment andAccreditation of Laboratory Animal Care, International and adheres toprinciples stated in the Guide for the Care and Use of LaboratoryAnimals, National Research Council, 2011.

TABLE 5 Therapeutic efficacy of mAbs in a mouse model of EBOV infection.No. Average mAb Treatment survivors/ weight loss competition group^(#)total (%)* P value group PBS 2/10 8.8 N/A N/A 2G4 4/10 7.2 0.095 KZ52competitor ADI-15956 7/10 8.6 0.008 HR2 PE-16 7/10 9.1 0.008 HR2ADI-15974 8/10 8.0 0.002 HR2 ADI-15758 8/10 9.8 0.009 HR2 ADI-1599910/10  8.1 <0.0001 HR2 ADI-15820 6/10 9.9 0.026 HR2 ADI-15848 7/10 8.20.008 HR2 ADI-15960 5/10 15.4 0.073 Undefined ADI-15903 4/10 8.4 0.079Undefined ADI-15959 5/10 8.4 0.040 Undefined ADI-15765 4/10 5.9 0.102Undefined ADI-15818 3/10 13.8 0.164 KZ52 competitor PE-87 8/10 10.40.005 KZ52 competitor ADI-15734 6/10 7.3 0.026 KZ52 competitor ADI-157848/10 9.1 0.002 KZ52 competitor ADI-15772 7/10 11.8 0.011 KZ52 competitorPE-24 10/10  10.5 0.0003 KZ52 competitor ADI-15731 4/10 9.2 0.059 13C6competitor ADI-15932 4/10 12.1 0.139 13C6 competitor ADI-15940 4/10 11.00.059 13C6 competitor ADI-15744 4/10 12.3 0.095 13C6 competitorADI-16037 8/10 7.0 0.005 13C6 competitor ADI-16044 2/10 9.9 0.263 13C6competitor ADI-15817 4/10 9.9 0.095 13C6 competitor PE-47 9/10 10.10.006 Undefined PE-05 9/10 8.2 0.008 Undefined ^(#)Mice were given 100υg of the indicated antibody, or PBS, two days post infection. *Averageweight change from the pre-injection baseline to the peak of clinicaldisease. Mice were weighed as groups.

SUDV Challenge Studies in Mice

4-5 week old, IFNa/bR KO mice will be inoculated I.P. with SUDV (1000pfu). Experimental group will be treated with mAbs (0.3 ml volume) atindicated dose on days 1 and 4 post-infection. Control mice will vehiclecontrol I.P. (0.3 ml volume) on the same schedule as experimental mice.Mice will be observed daily for 21 days for moribund condition. Moribundmice will be promptly euthanized (IAW SOP AC-11-07) when they meeteuthanasia criteria (score sheet).

Reagents and Animals Required:

Number # of Treat- Dose of Animals to Challenge Challenge Group ment(ug) Animals Challenge Dose Virus Grp 1 PE-87 300 10 10 1000 pfu SUDVGrp 2 PE-24 300 10 10 1000 pfu SUDV Grp 3 PE-16 300 10 10 1000 pfu SUDVGrp 4 PBS n/a 10 10 1000 pfu SUDV Total 40 40Species: IFNa/bR KO; Number of pans: 4; Days Required: 21; mAb: 300ug/dose (20 mg/kg): 300 ul of stock mAb per mouse

Time Line Day Date Task 0 4 Feb. 2016 Challenge I.P. 1 5 Feb. 2016 TreatI.P. with 300 ul per mouse 4 8 Feb. 2016 Treat I.P. with 300 ul permouse 21 25 Feb. 2016 Terminate Study

Materials and Methods 2 Cells

Vero African grivet monkey cells and 293T human embryonic kidneyfibroblast cells were maintained in high-glucose Dulbecco's modifiedEagle medium (DMEM; Thermo Fisher) supplemented with 10% fetal bovineserum (Atlanta Biologicals), 1% GlutaMAX (Thermo Fisher), and 1%penicillin-streptomycin (Thermo Fisher). Cells were maintained in ahumidified 37° C., 5% CO2 incubator.

Vesicular Stomatitis Virus (VSV) Recombinants and Pseudotypes

Recombinant vesicular stomatitis Indiana viruses (rVSV) expressing eGFPin the first position, and encoding representative GP proteins fromEBOV/Mayinga (EBOV/H.sap-tc/COD/76/Yambuku-Mayinga), EBOV/Makona(EBOV/H.sap-rec/LBR/14/Makona-L2014), BDBV(BDBV/H.sap/UGA/07/But-811250), SUDV/Boneface(SUDV/C.por-lab/SSD/76/Boneface), RESTV(RESTV/M.fas-tc/USA/89/Phi89-AZ-1435), and LLOV(LLOV/M.sch-wt/ESP/03/Asturias-Bat86), in place of VSV G have beendescribed previously [1-3]. VSV pseudotypes bearing eGFP and GP proteinsfrom TAFV (TAFV/H.sap-tc/CIV/94/CDC807212) and MARV(MARV/H.sap-tc/KEN/80/Mt. Elgon-Musoke) were generated as described [4].

Generation of cleaved VSV-GP particles and GPΔTM ectodomain proteins

In some experiments, cleaved viral particles bearing GP_(CL) were firstgenerated by incubation with thermolysin (200 μg/mL, pH 7.5, 37° C. for1 h; Sigma-Aldrich) or recombinant human cathepsin L (CatL, 2 ng/μL, pH5.5, 37° C. for 1 h; R&D Systems), as described previously [1].Reactions were stopped by removal onto ice and addition ofphosphoramidon (1 mM) or E-64 (10 μM), respectively, and viral particleswere used immediately for infectivity assays. A recombinant, solubleGPΔTM protein [5] was also essentially as described above.

VSV Infectivity Measurements and Neutralization Assays

Viral infectivities were measured by automated counting of eGFP+ cells(infectious units; IU) using a CellInsight CX5 imager (Thermo Fisher) at12-14 h post-infection. For mAb neutralization experiments, pre-titratedamounts of VSV-GP particles (MOI≈1 IU per cell) were incubated withincreasing concentrations of test mAb at room temp for 1 h, and thenadded to confluent cell monolayers in 96-well plates. Viralneutralization data were subjected to nonlinear regression analysis toderive EC₅₀ values (4-parameter, variable slope sigmoidal dose-responseequation; GraphPad Prism).

TABLE 6 rVSV-GP neutralization IC50 (nM)¹ mAb EBOV BDBV TAF SUDV RESTVPE-16 0.2 0.6 4.8 —² — PE-05 9.0 4.0 0.8 34 22 PE-24 2.0 1.0 1.5 0.5 0.2PE-87 0.5 0.5 1.0 0.3 0.2 PE-64 2.5 0.4 8 40 — ¹IC₅₀ (nM), mAbconcentration that affords half-maximal neutralization of viralinfectivity. ²No detectable neutralizing activity.

Authentic Filoviruses and Microneutralization Assays

The authentic filoviruses EBOV/“Zaire 1995”(EBOV/H.sap-tc/COD/95/Kik-9510621) [6], mouse-adapted EBOV/Mayinga(EBOV-MA) [7], SUDV/Boneface-USAMRIIDI11808, and BDBV/200706291 [8] wereused in this study. Antibodies were diluted to indicated concentrationsin culture media and incubated with virus for 1 h. Vero E6 cells wereexposed to antibody/virus inoculum at an MOI of 0.2 (EBOV, BDBV) or 0.5(SUDV) plaque-forming unit (PFU)/cell for 1 h. Antibody/virus inoculumwas then removed and fresh culture media was added. At 48 hpost-infection, cells were fixed, and infected cells were immunostainedand quantitated by automated fluorescence microscopy, as described [3].

TABLE 7 Authentic virus neutralization IC50 (nM)¹ mAb EBOV BDBV SUDVPE-16 0.1 0.3 300 PE-05 5.2 —² — PE-24 0.7 0.6 0.2 PE-87 0.2 0.6 0.2PE-64 0.6 1.5 120 ¹IC₅₀ (nM), mAb concentration that affordshalf-maximal neutralization of viral infectivity. ²No detectableneutralizing activity.Generation of mAbs

Recombinant mAbs from the human EBOV disease survivor, as well asgermline-reverted (IGL) mAb constructs and WT:IGL chimeras of PE-87 wereexpressed in Saccharomyces cerevisiae and purified from cellsupernatants by protein A affinity chromatography, as describedpreviously [5]. Other recombinant mAbs were produced in 293F cells bytransient transfection, and purified by protein A affinitychromatography, as described previously [3].

ELISAs for GP:mAb Binding

To identify GP cross-reactive mAbs, normalized amounts of rVSVs bearingEBOV, BDBV, and SUDV GP were coated onto plates at 4′C. Plates were thenblocked with PBS containing 3% bovine serum albumin (PBSA), andincubated with dilutions of test antibody (5, 50 nM). Bound Abs weredetected with anti-human IgG conjugated to horseradish peroxidase (SantaCruz Biotechnology) and Ultra-TMB colorimetric substrate (ThermoFisher). All incubations were performed for 1 h at 37° C.

Competition ELISAs for GP/mAb Binding to NPC1

The viral lipid envelopes of rVSV-EBOV GP particles were labeled withbiotin using a function-spacer-lipid construct (FSL-biotin)(Sigma-Aldrich) for 1 h at pH 7.5 and 37° C., as described [2].Biotinylated viral particles bearing GP_(CL) were generated byincubation with thermolysin, and then captured onto high-binding 96-wellELISA plates precoated with recombinant streptavidin (0.65 μg/mL;Sigma-Aldrich). Plates were then blocked with PBSA, and incubated withserial dilutions of test mAbs. Washed plates were then incubated with apre-titrated concentration of soluble, FLAG epitope-tagged, NPC1 domainC (NPC1-C) protein [9], and bound NPC1-C was detected with an anti-FLAGantibody conjugated to horseradish peroxidase (Sigma-Aldrich). Allincubations were performed for 1 h at 37° C.

ELISAs and Immunoblots to Detect mAb Inhibition of GP Cleavage

We used exposure of the NPC1-binding site in EBOV GP_(CL) as a proxy forsuccessful GP→GP_(CL) cleavage by CatL. rVSV-EBOV GP particles,biotinylated as above, were preincubated with mixtures of test mAb andirrelevant human IgG (test mAb at 50, 250, or 1000 nM; 1000 nM total IgGper reaction) for 1 h at pH 5.5 and 37° C. Reactions were then incubatedwith CatL (4 ng/μL and 37° C. for 30 min). Reactions were then stoppedwith E-64, readjusted to neutral pH with PBS, and captured ontostreptavidin-coated ELISA plates. NPC1-C binding was measured as above.

Samples treated with the highest concentration of test mAb were alsosubjected to western blotting. Cleaved GP1 species were detected byimmunoblotting with h21D10 mAb (a gift from Dr. Javad Aman) directlyconjugated to horseradish peroxidase.

Selection of Viral Neutralization Escape Mutants

Escape mutant selections were performed by serial passage of rVSV-GPparticles in the presence of test mAb. Briefly, serial 3-fold dilutionsof virus were preincubated with a concentration of mAb corresponding tothe IC₉₀ value derived from neutralization assays, and then added toconfluent monolayers of Vero cells in 12-well plates, in duplicate.Infection was allowed to proceed to completion (>90% cell death by eye),and supernatants were harvested from the infected wells that receivedthe highest dilution (i.e., the least amount) of viral inoculum.Following three subsequent passages under mAb selection withvirus-containing supernatants as above, supernatants from passage 4 weretested for viral neutralization escape. If resistance was evident,individual viral clones were plaque-purified on Vero cells, and their GPgene sequences were determined as described previously [1]. Thefollowing escape mutant selections were performed: PE-16 with rVSV-EBOVGP/Makona, PE-24 with rVSV-SUDV GP/Boneface, PE-05 withrVSV-EBOV/Mayinga, and PE-64 with rVSV-BDBVAMuc.

Single-Particle Electron Microscopy

Antibody Fabs and a EBOV GPΔTM ectodomain protein were prepared asdescribed previously [5], and incubated at a ratio of 10:1 (Fab:GP)overnight at 4′C. Complexes were then deposited onto a carbon-coatedcopper mesh grid, and stained with 1% uranyl formate. Samples wereimaged on a Tecnai F12 microscope using the automated image acquisitionsoftware Leginon [10]. Images were collected with a Tietz 4K CMOSdetector at 52,000× magnification, resulting in a final pixel size of2.05 Å at the specimen level. Images were automatically uploaded to andprocessed within our Appion database [11]. Individual complexes wereextracted from raw images using DoG Picker [12], binned by 2, and placedinto a stack. The stack was then subjected to reference-free 2Dclassification using MRA/MSA [13]. Class averages that did not respondto Fab:EBOV GPΔTM complexes were removed from all subsequent analyses. Asubset of 2D class averages was used to create an initial model usingcommon lines within EMAN2 [14]. The raw particle stack was then refinedagainst the initial model using EMAN2 to yield the final 3D volumes.UCSF Chimera was used for modeling and figure generation [15].

GP:mAb Kinetic Binding Binding Analysis by Biolayer Interferometry (BLI)

The OctetRed™ system (ForteBio, Pall LLC) was used to determine thebinding properties of different IgGs to various forms of EBOV GP.Anti-human Fc (AHC) capture sensors (ForteBio) were used for initial mAbloading at 25 mg/mL in 1× kinetics buffer (PBS supplemented with 0.002%Tween-20 and 1 mg/mL of BSA). Binding to GP was performed acrosstwo-fold serial dilutions of EBOV GPΔTM or GP_(CL). The baseline anddissociation steps were carried out in the 1× kinetics buffer as per theinstrument manufacturer's recommendations. For analysis of binding at pH5.5, a 1× pH 5.5 kinetics buffer (50 mM sodium citrate dihydrate[pH5.5], 150 mM sodium chloride, 0.002% Tween-20 and 1 mg/mL BSA) was usedin place of the PBS-based 1× kinetic buffer for all steps. For all ofthe kinetics experiments, a global data fitting to a 1:1 binding modelwas used to estimate values for the k_(on) (association rate constant),k_(off) (dissociation rate constant), and K_(D) (equilibriumdissociation constant).

TABLE 8 K_(D) and IC₅₀ values for PE-87 and PE-87 CDR-H3 mutationsLigand Analyte K_(D)(nM) EBOV¹ BDBV¹ TAFV¹ SUDV¹ RESTV¹ PE-87 GP/GP_(CL)<0.001 1.0 0.4 0.9 0.8 0.1 D99A² GP 74 ± 1  >500 nn >350 nn nn H100A² GP4.5 ± 0.2 1.0 0.4 0.8 1.0 0.2 R101A² GP 9.4 ± 0.3 3.2 0.4 1.9 1.5 0.2V102A² GP 4.0 ± 0.1 0.6 0.2 1.0 1.1 0.2 W103A² GP 1.3 ± 0.1 nn >200 >150nn nn G106A² GP .03 ± .01 0.8 0.4 0.8 0.9 0.2 Y107A² GP 30 ± 1  3.0 0.80.8 >350 1.3 H108A² GP  18 ± 0.4 3.2 0.6 2.2 2.1 0.5 F109A² GP 37 ± 1 4.2 0.3 2.8 2.1 0.4 D110A² GP 1.3 ± 0.3 2.0 0.9 2.1 1.5 0.4 Y111A² GP5.9 ± 0.3 0.8 0.4 1.1 0.8 0.1 ¹IC₅₀ (nM), mAb concentration that affordshalf-maximal neutralization of vira infectivity. ²Mutation in CDR3 ofPE87.

EBOV and SUDV Challenge Studies in Mice

10-12 week old female BALB/c mice (Jackson Labs) were challenged via theintraperitoneal (i.p.) route with EBOV-MA (100 PFU; ˜3,000 LD₅₀). Micewere treated i.p. 2 days post-challenge with PBS vehicle or 300 μg ofeach mAb (0.3 mL volume, =15 mg mAb/kg). Animals were observed daily forclinical signs of disease and lethality. Daily observations wereincreased to a minimum of twice daily while mice were exhibiting signsof disease. Moribund mice were humanely euthanized on the basis ofIACUC-approved criteria.

6-8 week old male and female Type 1 IFN α/β receptor knockout mice (Type1 IFNα/β R−/−) (Jackson Labs) were challenged with WT SUDV (1000 PFUi.p.). Animals were treated i.p. 1 and 4 days post-challenge with PBSvehicle or 300 μg (≈15 mg mAb/kg) per dose, and monitored and euthanizedas above.

TABLE 9 Activity in mouse models Mouse efficacy (% survival) mAb EBOVSUDV PE-05 90 — PE-87 80 100 PE-16 70  20 PE-47 90 100 PE-24 100 90-100EBOV mouse studies (n = 10-30) were performed by Dr. J. Dye or P. Glass(USAMRIID) with mAb dosing (5-20 mg/kg) two days post-infection; SUDVmouse studies (n = 10) were performed by Dr. Dye with 10-20 mg/kg of mAbdosed one and four days post-infection.

BDBV Challenge Studies in Ferrets

Six-month-old female ferrets (Mustela putorius furo) were challenged viathe intramuscular (i.m.) route with WT BDBV(BDBV/H.sap-tc/UGA/07/Butalya-811250; 1000 TCID₅₀ in 0.5 mL volume), asdescribed previously [16]. Animals were treated i.p. 3 and 6 dayspost-challenge with either PBS vehicle or 15 mg (day 3) and 10 mg (day6) of each mAb (2 mL volume/dose). Additionally, 1 mL blood was takenfrom each animal on days 0, 3, 6, 10, 14, 21, 28 days post-infection todetermine viral load, measure complete blood counts, and evaluatebiochemical markers. Animals were monitored twice daily for signs ofdisease during the course of the experiment.

Animal Welfare Statement

Murine challenge studies were conducted under IACUC-approved protocolsin compliance with the Animal Welfare Act, PHS Policy, and otherapplicable federal statutes and regulations relating to animals andexperiments involving animals. The dfacility where these studies wasconducted (USAMRIID) is accredited by the Association for Assessment andAccreditation of Laboratory Animal Care, International (AAALAC) andadhere to principles stated in the Guide for the Care and Use ofLaboratory Animals, National Research Council, 2011.

Ferret challenge studies were approved by the Animal Care Committee(ACC) of the Canadian Science Centre for Human and Animal Health(CSCHAH) in Winnipeg, Canada, in accordance with guidelines from theCanadian Council on Animal Care (CCAC).

Statistical Analysis

Dose-response neutralization curves were fit to a logistic equation bynonlinear regression analysis. 95% confidence intervals (95% CI) for theextracted IC₅₀ parameter were estimated under the assumption ofnormality. Analysis of survival curves was performed with the Mantel-Cox(log-rank) test. Statistical comparisons of viral titers were carriedout with an unpaired t-test. Testing level (alpha) was 0.05 for allstatistical tests. All analyses were carried out in GraphPad Prism.

REFERENCES FOR MATERIALS AND METHODS 2

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We claim:
 1. A composition for the treatment of Ebola, the composition comprising: a therapeutically effective combination of i. a first monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13, and affinity matured variants thereof, wherein said first monoclonal antibody or antigen binding fragment thereof has a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 72, a heavy chain CDR3 comprising SEQ ID NO: 73, a light chain CDR1 comprising SEQ ID NO: 74, a light chain CDR2 comprising SEQ ID NO: 75, and a light chain CDR3 comprising SEQ ID NO: 76 and amino acid sequences 90% identical thereto, and wherein the antigen to which the antigen binding fragment binds comprises Ebola glycoprotein; and ii. a pharmaceutically acceptable excipient or carrier.
 2. The composition of claim 1, wherein said first monoclonal antibody or antigen binding fragment thereof binds at least two species of Filovirus glycoprotein.
 3. The composition of claim 1, wherein the first monoclonal antibody or antigen binding fragment that binds to the Ebola glycoprotein antigen thereof comprises predominantly a single glycoform.
 4. The composition of claim 3, wherein the predominantly single glycoform is one of GnGn, G1/G2, and NaNa.
 5. The composition of claim 3, wherein the predominantly single glycoform substantially lacks at least one of fucose and xylose.
 6. The composition of claim 2 further comprising a second monoclonal antibody or antigen binding fragment thereof, wherein said second monoclonal antibody or antigen binding fragment thereof binds Ebola glycoprotein.
 7. A composition for the treatment of Ebola, the composition comprising: a therapeutically effective combination of i. a first monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13, and affinity matured variants thereof, wherein said first monoclonal antibody or antigen binding fragment thereof binds at least two species of Filovirus, and wherein the antigen to which the antigen binding fragment binds comprises Ebola glycoprotein; and ii. a pharmaceutically acceptable excipient or carrier.
 8. The composition of claim 7, wherein said first monoclonal antibody or antigen binding fragment thereof has a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 72, a heavy chain CDR3 comprising SEQ ID NO: 73, a light chain CDR1 comprising SEQ ID NO: 74, a light chain CDR2 comprising SEQ ID NO: 75, and a light chain CDR3 comprising SEQ ID NO: 76 and amino acid sequences 90% identical thereto.
 9. The composition of claim 7, wherein the first monoclonal antibody or antigen binding fragment that binds to the Ebola glycoprotein antigen thereof comprises predominantly a single glycoform.
 10. The composition of claim 9, wherein the predominantly single glycoform is one of GnGn, G1/G2, and NaNa.
 11. The composition of claim 9, wherein the predominantly single glycoform substantially lacks at least one of fucose and xylose.
 12. The composition of claim 7 further comprising a second monoclonal antibody or antigen binding fragment thereof, wherein said second monoclonal antibody or antigen binding fragment thereof binds the Ebola glycoprotein.
 13. A monoclonal antibody or antigen binding fragment thereof effective to treat Ebola comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13, and affinity matured variants thereof, wherein said first monoclonal antibody or antigen binding fragment thereof comprises predominantly a single glycoform that binds at least two species of Filovirus, and wherein the antigen to which the antigen binding fragment binds comprises Ebola glycoprotein.
 14. The monoclonal antibody or antigen binding fragment thereof of claim 13, wherein said first monoclonal antibody or antigen binding fragment thereof has a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 72, a heavy chain CDR3 comprising SEQ ID NO: 73, a light chain CDR1 comprising SEQ ID NO: 74, a light chain CDR2 comprising SEQ ID NO: 75, and a light chain CDR3 comprising SEQ ID NO: 76 and amino acid sequences 90% identical thereto.
 15. The monoclonal antibody or antigen binding fragment thereof of claim 13, wherein the predominantly single glycoform is one of GnGn, G1/G2, and NaNa.
 16. The monoclonal antibody or antigen binding fragment thereof of claim 13, wherein the predominantly single glycoform substantially lacks at least one of fucose and xylose. 